Biocatalyst Loading Research Articles

Optimization of immobilization conditions of Mucor miehei lipase onto Florisil via polysuccinimide spacer arm using response surface methodology and application of immobilized lipase in asymmetric acylation of 2-amino-1-phenylethanols

In this study, the immobilization of Mucor miehei lipase onto Florisil® support via polysuccinimide spacer arm was scrutinized by using a 3-factor and 3-level Box–Behnken design. The independent parameters were immobilization pH, immobilization time and initial lipase concentration and the response was the specific activity of immobilized lipase. A quadratic equation was used to explain the relationship between the response and independent parameters. After analysis of variance test, coefficient of determination and adjusted coefficient of determination values were estimated as 0.98 and 0.94, respectively. The optimal immobilization pH, immobilization time and initial lipase concentration were determined as 6.0, 7 h and 1.1 mg mL−1, respectively, after desirability analysis. The specific activity values for three individual experiments were observed as 25.88 ± 0.73, 26.06 ± 0.47 and 25.96 ± 0.52 U mg protein−1 under the optimized conditions. The hydrolytic activities of free and immobilized lipase preparations were characterized using p-nitrophenyl palmitate as substrate. The esterification activity of immobilized lipase preparation was evaluated by asymmetric acylation of 2-(methylamino)-1-phenylethanol, 2-(ethylamino)-1-phenylethanol, 2-(butylamino)-1-phenylethanol, and 2-(hexylamino)-1-phenylethanol with vinyl acetate. The acylation protocol was optimized in terms of the effects of initial water amount, reaction temperature, molar ratio of amino alcohol to vinyl acetate, biocatalyst loading, organic medium and kind of lipases used. The developed protocol provided a facile methodology for the preparation of enantiopure 2-amino-1-phenylethanols which may be used as potential new β-adrenergic receptor antagonists.

Efficient desymmetrization of 4,6-di-O-benzyl-myo-inositol by Lipozyme TL-IM

The enantioselective enzymatic desymmetrization of 4,6-di-O-benzyl-myo-inositol, a myo-inositol derivative, was effectively catalyzed by Thermomyces lanuginosus lipase (TL-IM). The product 1D-1-O-acetyl-4,6-di-O-benzyl-myo-inositol, a useful precursor to inositol phosphates, was obtained in excellent yield and enantiomeric excess. Through the investigation of the effects of solvent, biocatalyst load, and temperature, a more economical procedure resulted. The feasibility of biocatalyst reuse was also shown.

Enzymatic synthesis of 1,3-dicaproyglycerol by esterification of glycerol with capric acid in an organic solvent system

Abstract In this work, the esterification of glycerol with capric acid catalyzed by an immobilized form of a 1,3-positionally selective lipase ( Rhizomucor miehei ) showed to be effective for the synthesis of 1,3-dicaprin in n -heptane as the reaction medium. The effects of the reaction parameters were studied using an experimental factorial design of three factors and three levels with two central points. The selected experimental variables were amount of glycerol adsorbed on silica gel ( G ), biocatalyst load ( E ) and reaction temperature ( T ), and the response variables were total conversion of capric acid, acylglycerol fractions, selectivity and yield of dicaprin, and acyl migration reaction. The range of each parameter was selected as follows: G = 50–250 mg, E = 20–40 mg and T = 40–60 °C. At optimum conditions 73% capric acid conversion was achieved, with 76% dicaprin selectivity, and selectivity to the specific 1,3-dicaprin of 70% of total products. An adequate selection of the reaction conditions is necessary not only to maximize the conversion of capric acid, but also to minimize the acyl migration reaction and the generation of undesired products. Evidence of kinetically controlled enzymatic acyl migration from sn-3/sn-1 to sn-2 is presented.

ChemInform Abstract: Evaluation of Immobilized Enzymes for Industrial Applications

AbstractReview: 60 refs.

Immobilization of Candida cylindracea lipase on poly lactic acid, polyvinyl alcohol and chitosan based ternary blend film: Characterization, activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N2 adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.

Production of propyl gallate in nonaqueous medium using cell-associated tannase ofBacillus massiliensis: Effect of various parameters and statistical optimization

Enzymatic synthesis of propyl gallate in an organic solvent was studied using cell-associated tannase (E.C. 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as a biocatalyst. The influence of buffer pH and strength, water activity, temperature, biocatalyst loading, gallic acid concentration, and 1-propanol concentration was studied by the one-factor-at-a-time method. Subsequently, response surface methodology was applied based on a central composite design to determine the effects of three independent variables (biocatalyst loading, gallic acid concentration, and 1-propanol concentration) and their mutual interactions. A total of 20 experiments were conducted, and a statistical model was developed, which predicted the maximum propyl gallate yield of 20.28 μg/mL in the reaction mixture comprising 40.4 mg biocatalyst, 0.4 mM gallic acid, and 6.52 % (v/v) 1-propanol in 9.5 mL benzene at 30°C. The subsequent verification experiments established the validity of the model. Under optimal conditions, 25% conversion of gallic acid to propyl gallate was achieved on a molar basis. The absence of the need for enzyme purification and subsequent immobilization steps and good conversion efficiency makes this enzyme system an interesting one. Reports on the applications of bacterial whole cell systems for synthetic reactions in organic solvents are scarce, and perhaps this is the first report on bacterial cell-associated tannase-mediated esterification in a nonaqueous medium.

Enzymatic processing of renewable glycerol into value-added glycerol carbonate

Increased production of biodiesel has led to excess glycerol production worldwide, which has resulted in a significant drop in glycerol prices. Glycerol carbonate is a multifunctional compound used as chemical intermediates, solvents, additives and monomers. In this study, the enzymatic synthesis of glycerol carbonate from glycerol and a dialkyl carbonate was investigated. Glycerol carbonate was formed when reacting glycerol with dimethyl carbonate, diethyl carbonate or dibutyl carbonate in the presence of Candida antarctica lipase B (Novozym 435), using tert-butanol as a solvent. Nearly 100% glycerol conversion was reached after 12 h, with glycerol carbonate being the primary product. The effects of reaction parameters including solvent choice and biocatalyst loading were also examined. The highest activity was found at restricted water conditions and when using tert-butanol as a solvent.

Evaluation of immobilized enzymes for industrial applications

In contrast to the application of soluble enzymes in industry, immobilized enzymes often offer advantages in view of stability, volume specific biocatalyst loading, recyclability as well as simplified downstream processing. In this tutorial review the focus is set on the evaluation of immobilized enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial applications.

Investigation of steapsin lipase for kinetic resolution of secondary alcohols and synthesis of valuable acetates in non-aqueous reaction medium

Abstract In present study, the application of steapsin lipase (as a biocatalyst) was investigated for kinetic resolution of secondary alcohols (1-phenyl ethanol and their derivatives) using vinyl acetate as an activated acyl donor. The enzymatic protocol was optimized for various reaction parameters such as effect of the molar ratio, solvent, temperature, time and biocatalyst loading to obtain best reaction conditions. On optimization, developed enzymatic methodology provided considerable enantiomeric excess of the product (up to 92% ee) at 55 °C in n -hexane as a solvent. Furthermore using the developed protocol, synthesis of several industrially important acetates was successfully achieved with excellent yield (up to 99%). During acetate synthesis, the biocatalyst was remarkably reused for eight consecutive recycles without any significant loss in its catalytic activity. This revealed the good potential of steapsin lipase for application in organic solvents.

Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO(2)) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO(2). The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45°C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO(2) conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO(2) conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.

Kinetic resolution of (R/S)-propranolol (1-isopropylamino-3-(1-naphtoxy)-2-propanolol) catalyzed by immobilized preparations of Candida antarctica lipase B (CAL-B)

Enzyme preparations of Candida antarctica B lipase (CAL-B) - immobilized on Eupergit C and partially modified Eupergit C supports - were tested for kinetic resolution of (R/S)-propranolol, using vinyl acetate as acyl donor, and toluene as organic solvent. The effects of (R/S)-propranolol concentration, vinyl acetate concentration and biocatalyst loading on the esterification and resolution of propranolol were studied. Additionally, different types of immobilized lipase derivatives were also evaluated in terms of its selectivity on kinetic resolution of (R,S)-propranolol. These derivatives showed different enantiomeric ratios (E), with high enantiomeric ratios (E=57) with CAL-B immobilized on Eupergit C supports.

An efficient bioprocess for enzymatic production of l-menthol with high ratio of substrate to catalyst using whole cells of recombinant E. coli

A gene encoding an esterase of Bacillus subtilis ECU0554 previously isolated from soil was cloned and overexpressed in Escherichia coli BL21. The recombinant esterase (recBsE) showed the best enantioselectivity ( E > 100) towards dl-menthyl acetate, in contrast to dl-menthyl esters propionate and butyrate. A high ratio of substrate to catalyst (S/C-ratio, ≥50) was achieved in the kinetic resolution of dl-menthyl acetate by using whole cells of recombinant E. coli BL21. Some key parameters of the biocatalytic process, including amount of cosolvent, catalyst loading and substrate loading, were optimized. Compared with the process catalyzed by wild-type whole cells of B. subtilis ECU0554, the second-generation bioprocess using whole cells of recombinant E. coli BL21 afforded a 40-fold improvement in S/C-ratio and a 75-fold improvement in the volumetric productivity per biocatalyst loading. Moreover, the substrate loading was increased up to 200 g L −1 (∼1 M), the biocatalyst loading was reduced to 2.5 g L −1 and the space-time yield was improved from 54 g L −1 d −1 to 202 g L −1 d −1.

Lipase-catalyzed acidolysis of tripalmitin with capric acid in organic solvent medium: Analysis of the effect of experimental conditions through factorial design and analysis of multiple responses

The acidolysis of tripalmitin with capric acid catalyzed by an immobilized form of a 1,3-positionally selective lipase (Rhizomucor miehei) showed to be effective for the synthesis of structured lipids of the MLL and MLM type. The effects that reaction parameters such as substrate molar ratio (N), biocatalyst load (E), and reaction temperature (T) have on selected responses variables (i.e. total conversion of tripalmitin, selectivity and yield of the desired structured lipid, hydrolysis yield, and acyl migration importance), were evaluated by use of an experimental factorial design of three factors and three levels with two central points and with a confidence level of 95%. The range of each parameter was selected as follows: N=3–9, E=5–15wt%, T=50–70°C. The statistical analysis of results was addressed by use of both simple linear models and more complicated quadratic models using specific commercial software. The results obtained showed that a proper selection of reaction conditions is needed in order to maximize not only the yield of the desired structured lipid, but also to minimize the generation of hydrolysis and acyl migration by-products.

A green-by-design biocatalytic process for atorvastatin intermediate

The development of a green-by-design, two-step, three-enzyme process for the synthesis of a key intermediate in the manufacture of atorvastatin, the active ingredient of the cholesterol lowering drug Lipitor®, is described. The first step involves the biocatalytic reduction of ethyl-4-chloroacetoacetate using a ketoreductase (KRED) in combination with glucose and a NADP-dependent glucose dehydrogenase (GDH) for cofactor regeneration. The (S) ethyl-4-chloro-3-hydroxybutyrate product is obtained in 96% isolated yield and >99.5% e.e. In the second step, a halohydrin dehalogenase (HHDH) is employed to catalyse the replacement of the chloro substituent with cyano by reaction with HCN at neutral pH and ambient temperature. The natural enzymes were highly selective but exhibited productivities that were insufficient for large scale application. Consequently, in vitro enzyme evolution using gene shuffling technologies was employed to optimise their performance according to predefined criteria and process parameters. In the case of the HHDH reaction, this afforded a 2500-fold improvement in the volumetric productivity per biocatalyst loading. This enabled the economical and environmentally attractive production of the key hydroxynitrile intermediate. The overall process has an E factor (kg waste per kg product) of 5.8 when process water is not included, and 18 if included.

Response Surface Methodology as an Approach to Determine Optimal Activities of Lipase Entrapped in Sol–Gel Matrix Using Different Vegetable Oils

The conditions for maximization of the enzymatic activity of lipase entrapped in sol-gel matrix were determined for different vegetable oils using an experimental design. The effects of pH, temperature, and biocatalyst loading on lipase activity were verified using a central composite experimental design leading to a set of 13 assays and the surface response analysis. For canola oil and entrapped lipase, statistical analyses showed significant effects for pH and temperature and also the interactions between pH and temperature and temperature and biocatalyst loading. For the olive oil and entrapped lipase, it was verified that the pH was the only variable statistically significant. This study demonstrated that response surface analysis is a methodology appropriate for the maximization of the percentage of hydrolysis, as a function of pH, temperature, and lipase loading.

Improved activity and thermostability of ( S)-aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine

The evolution of a mesophilic aminotransferase, isolated from Athrobacter citreus, to a thermostable aminotransferase was accomplished via error-prone PCR. After three rounds of mutagenesis, a mutant was generated that decreased the biocatalyst loading 3-fold. This improved biocatalyst was engineered further and a new mutant was isolated that was capable of the same performance with 5-fold reduction in biocatalyst loading. Overall, the best mutant (#6) enabled a 3-fold reduction in biocatalyst loading, almost a 5-fold increase in product concentration, and a 5-fold reduction in process cycle time. Through these rounds of mutagenesis enzyme specific activity improved from 5.9 to 1582.8 IU/g with an overall improvement in product yield due to reduced biocatalyst loading. The new mutants were also able to operate at temperatures greater than 50 °C for an extended period of time. A simple cost model was developed to describe the impact of enzyme improvement on product cost.

Optimal biocatalyst loading in a fixed bed

The optimal distribution of biocatalyst in a fixed bed operating at steady state was determined to minimize the length of the bed for a fixed conversion of 95%. The distribution in terms of the biocatalyst loading on an inert support depends upon the axial distance from the bed entrance (continuous solution) as well as a set of dimensionless parameters that reflect the bed geometry, the bulk flow, reaction kinetics and diffusional characteristics. A mathematical model of the system with the following features was used to obtain the results: (1) convective mass transfer and dispersion in the bulk phase; (2) mass transfer from the bulk phase to the surface of the catalyst particle; and (3) simultaneous diffusion and chemical reaction in the catalyst particle with Michaelis-Menton kinetics and a reliable diffusion model (Zhao and DeLancey in Biotechnol Bioeng 64:434-441, 1999, 2000). The solution to the mathematical model was obtained with Mathematica utilizing the Runge Kutta 4-5 method. The dimensionless length resulting from the continuous solution was compared with the optimal length restricted to a uniform or constant cell loading across the entire bed. The maximum difference in the dimensionless length between the continuous and uniform solutions was determined to be 6.5%. The model was applied to published conversion data for the continuous production of ethanol that included cell loading (Taylor et al. in Biotechnol Prog 15:740-751, 2002). The data indicated a minimum production cost at a catalyst loading within 10% of the optimum predicted by the mathematical model. The production rate versus cell loading in most cases displayed a sufficiently broad optimum that the same (non-optimal) rate could be obtained at a significantly smaller loading such as a rate at 80% loading being equal to the rate at 20% loading. These results are particularly important because of the renewed interest in ethanol production (Novozymes and BBI International, Fuel ethanol: a technological evolution, 2004).

Enzymatic Conversion of Waste Cooking Oils Into Alternative Fuel-Biodiesel

Production of biodiesel from pure oils through chemical conversion may not be applicable to waste oils/fats. Therefore, enzymatic conversion using immobilized lipase based on Rhizopus orzyae is considered in this article. This article studies this technological process, focusing on optimization of several process parameters, including the molar ratio of methanol to waste oils, biocatalyst load, and adding method, reaction temperature, and water content. The results indicate that methanol/oils ratio of 4, immobilized lipase/oils of 30 wt% and 40 degrees C are suitable for waste oils under 1 atm. The irreversible inactivation of the lipase is presumed and a stepwise addition of methanol to reduce inactivation of immobilized lipases is proposed. Under the optimum conditions the yield of methyl esters is around 88-90%.

Biocatalytic membranes for ultrafiltration treatment of wastewater containing dyes

A possibility to prepare the biofunctional membranes showing the biocatalytic properties and use those in post-treatment of wastewater containing synthetic dyes have been established. Selected Pseudomonas mendocina and Bacillus subtilis cultures were used as biocatalysts for dye destruction. It has been established that cells in spore form are able to survive in N-methylpyrrolidone that allow to use method of polymer solution casting for membrane preparation. The optimal conditions for entrapping of whole cells of microorganisms into the polymer matrix have been determined. Membrane biocatalytic activity has been studied depending on method of casting solution preparation, biocatalyst loading and operating parameters. Dye destruction occurs both in membrane pores and on membrane surface. Membrane obtained provide discolouring of treated solutions (permeate). The dye concentration in retentate depends on the trans-membrane fluxes. The concentration in retentate need not be observed at relatively low fluxes (up to 20 l/m2 h).

Bioconversion of trans-cinnamic acid to l-phenylalanine by l-phenylalanine ammonia-lyase of Rhodotorula glutinis: Parameters and kinetics

The kinetics of the bioconversion of trans-cinnamic acid to l-phenylalanine by the l-phenylalanine ammonialyase (PAL) enzyme of resting cells of Rhodotorula glutinis have been studied under the optimum bioreaction conditions determined. The optimum pH, temperature, ammonia concentration, and biocatalyst loading were 10.5, 30°C, 8 m, and ca. 2 (w/w), respectively. Among various chemicals, sodium glutamate and penicillin were found to increase the PAL activity of the cells. A maximum concentration of 76.18 m m (12.57 g dm −3) l-phenylalanine was maintained from 100 m m (14.8 g dm −3) trans-cinnamic acid after 104 h of residence time in the fed-batch operation of the bioreactor. trans-Cinnamic acid concentrations higher than 30–50 m m were shown to cause the substrate inhibition, besides the mixed-type inhibition effect of the chloride ions. Mechanistic bioreaction rate equations that incorporate separately the inhibition effects of the trans-cinnamic acid and chloride ions, and the activation effect of sodium glutamate, were proposed and the kinetic parameters of the models were calculated.